1) The kinetics of inhibition of active transport caused by colicin E1 will be studied along with trypsin rescue during inhibition, the release of labeled colicin fragments, and the change in electrochemical potential during the period (less than 1 min.) of the fast inhibition, with the purpose of determining the extent of colicin penetration during the early part of inhibition. 2) We will also ask whether rapid inhibition is a consequence of a steep power dependence of active transport on electrochemical potential. 3) The rate of entry of colicin into the cell envelope would be measured using a fluorescence probe bound to colicin E3, and a differential fluourescence polarization assay. 4) The rapid inhibition of active transport also implies that utilization of intermembrane connections by colicin-receptor complexes is very efficient. The role of such connections, which are possibly "adhesion zones", will be studied as a function of cell plasmolysis. High osmotic strength confers insensitivity to E1. Membrane connections will be assayed by phage BF23 sensitivity, and outer membrane fluorescence probe response occurring upon deenergization of inner membrane. 5) The role of H ion movement in membrane energization will be further studied in membrane vesicles, where comparative measurements will be made of the rates of H ion efflux, O2 consumption, and oxidation of respiratory components. 6) Studies on changes in colicin properties upon insertion into membranes require colicin preparations of optimal activity. Preparations of colicin E2 with high activity would be studed, with very active preparations now obtained by gel filtration, and preparations of conventional activity by hydrophobic chromatography. 7) Single component (DMPC or DPPC) liposome preparations have been characterized with respect to their phase transitions. It is planned to incorporate colicin E1 into liposomes to measure specific ion conductance changes. Characterization of the phase transition with a fluorescence probe and differential scanning calorimetry before and after addition of colicin will allow the conductance to be measured as a function of bulk lipid phase and determination of colicin channel properties. Controls will be done with other basic proteins such as lysozyme or cytochrome c. If colicin can be effectively incorporated into liposomes, it is planned to study structural changes of colicin using circular dichroism.